Background: In hereditary non-polyposis colorectal cancer, over 90% of the identified mutations are in two genes, hMSH2 and hMLH1. A large proportion of the mutations detected in these genes are of the missense type which may be either deleterious mutations or harmless polymorphisms. Aim: To investigate whether nine missense and one splice site mutation of hMLH1 and hMSH2, in 10 kindreds with a familial history of colorectal cancer or young age of onset, could be interpreted as pathogenic. Methods: Clinical and genetic characteristics were collected: (i) evolutionary conservation of the codon involved; (ii) type of amino acid change; (iii) occurrence of mutation in healthy controls; (iv) cosegregation of mutation with disease phenotype; (v) functional consequences of gene variant; and (vi) microssatellite instability and immunoexpression of hMSH2 and hMLH1 analysis. Results: Seven different missense and one splice site mutation were identified. Only 1/8 was found in the control group, 2/7 occurred in conserved residues, and 5/7 resulted in non-conservative changes. Functional studies were available for only 2/8 mutations. Segregation of the missense variant with disease phenotype was observed in three kindreds. Conclusion: In the majority of families included, there was no definitive evidence that the missense or splice site alterations were causally associated with an increased risk of developing colorectal cancer. Until further evidence is available, these mutational events should be regarded and interpreted carefully and genetic diagnosis should not be offered to these kindreds.

Mutation searching was performed in the hMSH2 and hMLH1 genes in 20 Portuguese families representing 124 registered affected individuals. Of the 20, 16 fulfilled the classic 'Amsterdam' criteria for HNPCC, whereas the remaining four families satisfied a modified set of criteria. These criteria required a CRC diagnosed before age 50 years and cancers diagnosed in two other relatives within the HNPCC spectrum. A multi-method approach was performed using the protein truncation test (PTT), single strand conformation polymorphism (SSCP) with two different sets of conditions, heteroduplex analysis (HA) and denaturing gradient gel electrophoresis (DGGE). Putative phenotype-genotype correlations were also explored. Ten different germline mutations were identified. Six of these were found in hMLH1 in seven families and four in hMSH2 in four families. SSCP and DGGE had the highest diagnostic yields with the percentage of variants detected above 67% and together HA and PTT had the lowest. No single technique detected all variants. Trends for the absence of extracolonic manifestations were observed in families carrying hMLH1 germline mutations (four of seven in hMLH1 vs one of four in hMSH2). Most of the families with rectal cancer were associated with hMLH1 (six of seven in hMLH1 vs two of four in hMSH2). A multi-technique approach is necessary to identify a high percentage of germline mutations. Seven novel mutations were found in this Portuguese population.

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BACKGROUND Hereditary nonpolyposis colorectal carcinoma (HNPCC) significantly raises the risk of developing colorectal carcinoma (CRC) and other extracolonic tumors. It is defined by the Amsterdam Criteria and is associated with germline mutations in mismatch repair genes, primarily MLH1 and MSH2. The objectives of the current study were to evaluate the presence of CRC (Type I) and other extracolonic tumors (Type II) in families with HNPCC and to analyze the findings for correlations with germline mutations in the MLH1 and MSH2 genes. METHODS Seventy families with an HNPCC diagnosis were analyzed. Denaturing gradient gel electrophoresis and direct sequencing were used for germline mutation analysis in the MLH1 and MSH2 genes. RESULTS Forty‐three of 70 families (61%) presented with HNPCC Type II. In 21 of 30 families that had a complete genetic diagnosis, 16 pathogenic germline mutations (7 MLH1 mutations and 9 MSH2 mutations) and 5 mutations of unknown pathogenecity (all MLH1 mutations) were found. In the remaining nine families, no mutations were detected. Unequivocally pathogenic mutations were far more common in families with HNPCC Type II compared with families that had CRC only (P = 0.01). Families with endometrial carcinoma presented with the greatest probability of mutational detection (P = 0.005). MLH1 was only gene affected in families with HNPCC Type I, whereas mutations in both MLH1 and MSH2 were found in families with HNPCC Type II (P = 0.04). However, the MSH2 gene was more frequently involved in families with HNPCC in which endometrial carcinoma was present (P = 0.005). CONCLUSIONS CRC and endometrial carcinoma were associated with a greater probability of detecting pathogenic mutations in mismatch repair genes, with MSH2 involvement predominating. The results support specific mutational screening strategies, based on observed phenotypes, for families with HNPCC. Cancer 2004. © 2004 American Cancer Society.